PROJECT SUMMARY Viral infections due to cytomegalovirus (CMV), Epstein-Barr virus (EBV), adenovirus (Ad) and BK virus (BKV) remain a significant cause of treatment failure in patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). Individuals living with HIV who require a transplant for an underlying malignancy are also at high risk for viral rebound. Recipients of cord blood (CB) or HSC from virus-nave donors are at particular risk since their grafts contain no virus-specific memory T-cells. Antiviral drugs are effective only for some viruses, and most have significant toxicities. Adoptive transfer of virus-specific cytotoxic T lymphocytes (VSTs) from the stem cell donor has proved safe and highly effective, but has generally only been for recipients of grafts from virus-experienced donors, thereby excluding patients at highest risk. This lack of an effective strategy to activate and expand virus-specific T-cells from nave donor sources such as CB, has been a major obstacle to extending the approach to high-risk recipients, whose continued prolonged morbidity and high mortality from viral diseases substantially reduces the cost:benefit ratio of the transplant procedure. In the last funding cycle, we developed a novel approach that effectively expanded VSTs specific for (CMV), (EBV) and (Ad) from nave CB-derived T-cells. CB-VSTs targeting three viruses had broad epitope specificity, were safe and effectively prevented and/or treated viral infections in 12 pediatric patients after single cord blood transplantation (CBT). To broaden the applicability of this approach, we now propose studies to: (i) extend this approach to additional viruses (e.g. BKV and HIV), (ii) employ a more rapid manufacturing protocol and (iii) evaluate VST therapy in adult patients after double unrelated CBT (DUCBT). Hence, we now hypothesize that, following CBT, the infusion of rapidly manufactured CB-derived VSTs targeting FOUR viruses (CMV, EBV, Ad, BKV) will be safe (Aim 1) and provide broad protection early (< 60 days) against viral infections arising post- DUCBT (Aim 2). We further hypothesize that this approach will be effective for the priming of HIV-specific T- cells from CB, potentially as a curative strategy post-CBT (Aim 3). The results we generate will show whether this novel strategy can consistently produce effective VSTs directed to the commonest pathogenic viruses after CBT, including HIV, and whether this approach has the potential to reduce morbidity and mortality after transplantion. Our demonstration, during the last funding cycle, of the safety and feasibility of adoptive transfer of VSTs to pediatric patients undergoing CBT set the stage for collaborations with Projects 1 and 3 (longitudinal T-cell reconstitution in clinical trials of CB expansion/homing and of CB-NK cells, repectively and fucosylation) as well as Project 4, whose chimeric antigen receptor targeting approach could be used to genetically modify VSTs to render them specific for conventional virus epitopes.